The long-term objective is to use animal models to find mechanisms and approaches whereby adoptively transferred T cells transduced with mutant neoantigen-specific TCRs can eradicate solid cancers in patients without destroying normal tissues. Tumors in patients have been present at least for months or years and are usually at least 1cm in diameter at time of diagnosis. Therefore, large and long-established solid tumors expressing autochthonous (untransfected) mutant neoantigens are the focus of this proposal. A key element being explored is the destruction of the immunosuppressive stromal tumor microenvironment by transfer of MHC Class II restricted neoantigen-specific TCR-transduced CD4+ T cells (CD4+TCR). These T cells cause tumor destruction followed by long-term arrest of tumor growth. A second key element being explored is that CD4+TCR T cells form 4-cell-type clusters with MHC Class I restricted neoantigen-specific TCR-transduced CD8+ T cells (CD8+TCR) which are attracted to the cluster by recognizing the cross-presented neoantigen on stromal CD11b+ tumor-associated macrophages. Finally, intraclonal non-heritable heterogeneity is explored as novel mechanism of escape of cancers despite cancer cells seemingly lacking mutant neoantigen-negative variants. Aim 1 is to determine the requirements and mechanisms for CD4+TCR T cells to destroy, without CD8+ T cells, advanced solid tumors and subsequently arrest their growth. It will be tested whether targeting tumor stroma is essential for this CD4+TCR-mediated destruction/arrest and whether these effects are independent of direct cancer cell recognition. Furthermore, it will be examined whether therapeutic CD4+TCRs can be isolated from tumor-infiltrating lymphocytes (TILs) that are failing to reject the cancer. Finally, it will be tested whether appropriate lysosomal processing of mutant neoantigen is a decisive predictor for CD4+TCR- mediated tumor destruction. Aim 2 is to determine whether 4-cell-type clusters at the effector phase are essential for cancer eradication by CD4+TCRs and CD8+TCRs. It will be analyzed whether direct cancer cell recognition by the CD8+TCR is required for cancer eradication. Furthermore, it will be tested whether, for synergistic cancer cell destruction, the two neoantigens must be released from the same cancer cell because this would allow the CD8+TCR T cells to recognize the cross-presented neoantigen on the same CD11b+ stromal cell also being recognized by the CD4+TCR T cells. Antigen-specific 4-cell-type cluster formation will be quantified and modeled in vitro using longitudinal time-lapse imaging. Finally, will be determined whether cancers brought into equilibrium by CD4+TCRs can still be eradicated by subsequent treatment with CD8+TCR transduced T cells. Aim 3 is to determine mechanisms of escape and the molecular nature of CD8+TCR recognized antigens that can be used to eradicate cancers in synergy with CD4+TCRs. It will be examined whether cloned cancer cells seemingly lacking antigen-negative variants nevertheless escape by the presence of cancer cells with low mutant mRNA expression due to intraclonal heterogeneity.